Genetic engineering methods are increasingly allowing precursor forms of insulin to be expressed in microorganisms (EP-A-347 781, EP-A-367 163). The pre pro sequences are usually cleaved off chemically and/or enzymatically (DE-P-3 440 988, EP-A-0264250). Known enzymatic conversion methods are based on cleavage with trypsin and carboxypeptidase B (Kemmler W. et al. J. Biol. Chem., 246 (1971) 6786-6791; EP-A-195 691; EP-B-89007). In the typical process of conversion of Insulin precursor molecule to the corresponding molecule, the linker peptide between the A and B chains are removed. The enzymatic reactions with trypsin is an enzymatic and complex reaction that cleaves not only those peptide bonds whose cleavage produces human insulin or the desired end products but also, in a competing reaction, cleavage at other susceptible sites produce a plurality of undesired byproducts.
Prior art methods include the use of trypsin and a second enzyme carboxypeptidase in such manner that the second enzyme carboxypeptidase is added when the required intermediate will be formed in the reaction. The disadvantage of these methods is the formation of large amounts of impurity byproducts which can be removed from the reaction solution only with difficulty. In the particular case of the conversion of human precursor form of into human insulin (human insulin, HI), there is formation of large amounts of des-Thr(B30)-human insulin (des-Thr(B30)-HI).
From the above, it is evident that it would be advantageous to follow a simpler enzymatic reaction process for cleavage of precursor insulin compounds, their analogs and derivatives thereof to insulin through a much simpler step that removes the possible formations of polymeric impurities as completely as possible and, at the same time, increases the concentration of desired insulin end product as much as possible. An additional condition is the need to ensure a high yield overall enhancing the ease of operation, quality as well as the quantity of the desired end product.
The disadvantages of the known processes have been remedied by the enzymatic reactions carried out in the instant invention, and it has turned out that the increase in yield without undesired byproducts is relative to the optimal concentration of trypsin and carboxypeptidase used under conducive reaction conditions. The inventors have endeavored to develop an improved one step enzymatic reaction process involving the combinatorial and concurrent use of optimal quantities of trypsin and carboxypeptidase B that work synergistically to provide the desired end products enhancing the ease of operation, purity and yield of the end products.